3-[(S)-1&#39;-phenylethylamino]propylaminobleomycin, non-toxic salt thereof, and method for producing same

ABSTRACT

Novel 3-[(S)-1&#39;-phenylethylamino]propylaminobleomycin obtained by reacting a reactive derivative of the carboxyl group of bleomycinic acid with N-[(S)-1&#39;-phenylethyl]-1,3-diaminopropane, a non-toxic salt of said novel bleomycin, and a method for producing the novel bleomycin. Because of much reduction in the side effect causing pulmonary fibrosis, the novel bleomycin is more useful than a commercial bleomycin complex which gives rise to said undesirable side effect.

This is a division of application Ser. No. 915,805 filed June 15, 1978.

This invention relates to a novel bleomycin, non-toxic salts thereof,and a method for producing said novel bleomycin. More particularly, itrelates to 3-[(S)-1'-phenylethylamino]propylaminobleomycin representedby the formula ##STR1## non-toxic salts thereof, and a method forproducing said novel bleomycin.

Bleomycins are carcinostatic antibiotic substances discovered byUmezawa, one of the present inventors, and collaborators [Journal ofAntibiotics, 19A, 200 (1966)], which are water-soluble basicglycopeptides produced by Actinomycete Streptomyces verticillus and arecapable of readily chelating one atom of divalent copper. In normalcultivation, 16 components of bleomycins are produced and isolated [forexample, Umezawa et al., Journal of Antibiotics, 19A, 210 (1966)]. Ofthe bleomycins, a mixture of copper-free A₁, A₂, A₅, B₂ and demethyl A₂(hereinafter referred to as "Bleomycin complex") has been widely used inclinical fields of cancer therapy and has proved to be successfulparticularly in the therapy of squamous cell carcinoma as major target,skin cancer, head and neck cancer, cancer of the uterine cervix, lungcancer, and malignant lymphoma.

Regarding the side effect, however, there have been reported cases ofpumonary fibrosis and other undesirable occurences. Of the side effectsexhibited by bloemycins, the most fearful is pulmonary fibrosis. It isreadily imaginable that the carcinostatic activity of bleomycins mightbe more effectively manifested in clinical fields if the above-notedside effect could be more or less reduced.

Under the circumstances, the present inventors had engaged for years inthe synthesis of various bleomycins and in various animal tests on theircarcinostatic activity as well as their toxicity including pulmonaryfibrosis as major test item. As a result, it was found that3-[(S)-1'-phenylethylamino]propylaminobleomycin (hereinafter referred toas "NK631" including both copper-containing and copper-free forms),which is a new bleomycin obtained by reacting a reactive derivative ofthe carboxyl group of bleomycinic acid withN-[(S)-1'-phenylethyl]-1,3-diaminopropane, is markedly reduced in theside effect causing pulmonary fibrosis and is not deteriorated incarcinostatic activity, as compared with a commercial bleomycin complexand other known bleomycins. The present invention has been accomplishedbased on the above finding.

An object of this invention is to provide a novel bleomycin,3-[(S)-1'-phenylethylamino]propylaminobleomycin, a non-toxic saltthereof, and a method for producing said novel bleomycin.

Other objects and advantages of this invention will become apparent fromthe following description.

The excellent bioactivity of the present bleomycin represented by theformula (I) is illustrated below with reference to ExperimentalExamples.

The bioactivity of NK631 was examined with respect to the following 4test items by comparing with a commercial bleomycin complex and3-[(R,S)-1'-phenylethylamino]propylaminobleomycin monosulfate(copper-free form) (hereinafter referred to as "RS form"). The RS formwas obtained by a fermentation procedure disclosed in U.S. Pat. No.3,846,400.

1. Pulmonary fibrosis

2. Anti-tumor activity

3. Antimicrobial activity

4. Toxicity

1. Toxicity to the lung of mice (fibrosis)

ICR strain mice (male, 15 weeks old), 12 in number per group, were used.The dosage of each pharmaceutical test preparation was 5 mg/kg. The dosewas administered by intraperitoneal injection, once a day, for 10consecutive days. After administration, the mice were bred for 5 weeks.After observation, the mice were slaughtered and autopsied to determinethe extent of pulmonary fibrosis. The incidence and grade of pulmonaryfibrosis in mice administered with the novel bleomycin of thisinvention, a commercial bleomycin complex and RS form were compared. Theresults obtained were as shown in Table 1.

                                      Table 1                                     __________________________________________________________________________               Incidence                                                                     Number of mice Grade                                                          with pulmonary Total score of pulmonary*                                      fibrosis Relative                                                                            fibrosis/total number of                                                                    Relative                                         (%)      value samples       value                                 __________________________________________________________________________    NK631 monosulfate                                                                         4/12 (0.33)                                                                           0.36   6/36 (0.17)  0.25                                  (copper-free form)                                                            RS form     7/12 (0.58)                                                                           0.63  12/36 (0.33)  0.49                                  Bleomycin complex                                                                        11/12 (0.92)                                                                           1.0   24/36 (0.67)  1.0                                   __________________________________________________________________________     Note:                                                                         *0 point: No fibrosis                                                         1 point: Accumulation of exudate in aveolus and fibrosislike change in        alveolar septum                                                               2 points: Fibrosis in several areas                                           4 points: Scattered fibrosis                                                  6 points: Fibrosis in more than twothirds of the total area              

As is apparent from the above results, as compared with the cases of RSform and bleomycin complex, the fibrosis due to NK631 monosulfate(copper-free form) was reduced to about 1/2 and 1/3, respectively, inincidence and about 1/2 and 1/4, respectively, in grade, indicating theusefulness of NK631 monosulfate in clinical fields.

2. Anti-tumor activity

2-1. Action on cultivated HeLa S₃ cell

ID₅₀ for each bleomycin was calculated from the percentage growthinhibition in 72 hours of culture in the presence of each bleomycin.ID₅₀ for NK631 was found to be 0.82 mcg/ml, as contrasted to 1.70 mcg/mlfor Bleomycin complex, indicating that NK631 inhibited the cell growthtwice as strongly as Bleomycin complex. ID₅₀ for RS form was found to be0.80 mcg/ml, indicating that the inhibiting effect was comparable tothat of NK631 monosulfate (copper-free form).

2-2. Carcinostatic activity to mouse Ehrlich cancer (solid tumor)

Each 2×10⁶ cells were transplanted subcutenously into the inguinalregion of ICR strain mice (male, 6 weeks old). After 24 hours, eachmouse was administered with test preparations in the same manner asdescribed in 1-2 above. On the 15th day after the subcutenoustransplantation, tumors were removed from each mouse and compared theweight with that of tumors developed in the control group, which hadreceived no treatment, to determine the percentage inhibition.

As shown in Table 2, the carcinostatic activity of NK631 monosulfate(copper-free) was comparable to that of the RS form and about 1.4 timesas high as that of the Bleomycin complex.

                  Table 2                                                         ______________________________________                                               Percentage inhibition                                                  Dosage   NK631 monosulfate       Bleomycin                                    mg/kg × 10                                                                       (Cu-free)     RS-form   complex                                      ______________________________________                                        2.7      84            78        67                                           0.9      68            60        55                                           0.3      39            51        49                                           0.1      24            51        24                                           0.03     19            13        21                                           0        0             0         0                                            ID.sub.50                                                                     mg/kg/day                                                                              0.35          0.31      0.49                                         ______________________________________                                    

2-3. Carcinostatic activity against ascites hepatoma (ascites type) inrats

Each 1×10⁶ AH66 cells were transplanted intraperitoneally into Donryustrain rats. After 24 hours, the test preparation was intraperitoneallyadministered once a day for 10 consecutive days. During a period of 30days after the transplantation, the weight of each rat and the number ofdead and survival were observed.

                  Table 3                                                         ______________________________________                                        Relative value of average survival                                            days                                                                          Dosage   NK631 monosulfate       Bleomycin                                    mg/kg × 10                                                                       (Cu-free)     RS-form   complex                                      ______________________________________                                        3.12     296           298       241                                          1.56     269           257       257                                          0.78     216           196       145                                          0.39     163           149       116                                          0.19     116           121       110                                          0        100           100       100                                          ______________________________________                                    

As shown in Table 3, in the activity against AH66 ascites hepatoma,NK631 monosulfate (copper-free form) was comparable to the RS-form andsuperior to the Bleomycin complex at every dosage.

2-4. Inhibitory activity against mouse squamous cell carcinoma inducedby 20-methylcholanthrene (hereinafter referred to as "20-MC")

An acetone solution of 20-MC was topically applied to the sheared backof ddy strain mice (male, 10 weeks old), twice a week, for 18 weeks.After 5 weeks from the beginning of the 20-MC treatment, 62.5 mcg/mouseof the test preparation was intraperitoneally applied, twice a week,during a period of 15 weeks. Within the first week after completion ofthe administration of test preparation, the region where 20-MC had beenapplied was pathologically examined for the incidence of carcinogenesis.

As shown in Table 4, NK631 monosulfate (copper-free form) and RS-formwere slightly more effective than the bleomycin complex in inhibitingthe carcinogenesis due to 20-MC.

                  Table 4                                                         ______________________________________                                                          Incidence of                                                                              Percentage                                                        carcinogenesis                                                                            inhibi-                                                   Mortality                                                                             (%)         tion                                            ______________________________________                                        NK631 monosulfate                                                                         1/12       5/11 (45.4)                                                                              47.8                                        (copper-free)                                                                 RS-form     1/12       5/11 (45.4)                                                                              47.8                                        Bleomycin complex                                                                         2/12       5/10 (50.0)                                                                              42.5                                        Control     1/24      20/23 (86.9)                                                                              0                                           ______________________________________                                    

As seen from the above results, NK631 monosulfate (copper-free form)clearly showed a superior anti-tumor activity compared with a commercialBleomycin complex.

3. Antimicrobial activity

The antimicrobial potency was assayed by the cup method againstMycobacterium 607 and Bacillus subtilis PCI by using bleomycin A₂ asstandard (1,000 U/mg). The results were as shown in Table 5.

                  Table 5                                                         ______________________________________                                                         M. 607   B. Sub.                                             ______________________________________                                        NK631 monosulfate (Cu-free)                                                                      7848       1550                                            RS-form            7535       1400                                            Bleomycin complex  1234       886                                             ______________________________________                                    

As is apparent from Table 5, NK631 monosulfate (copper-free form) andthe RS-form showed far superior antimicrobial potencies than that of theBleomycin complex.

4. Toxicity

4-1. Acute toxicity in intraperitoneal route (LD₅₀) in rats

As shown in Table 6, LD₅₀ value to rat in NK631 monosulfate (copper-freeform) which is approximately comparable to those for the Bleomycincomplex and RS-form.

                  Table 6                                                         ______________________________________                                                               Confidence limits                                                    LD.sub.50                                                                              (5% level of                                                         mg(w)/kg significance)                                          ______________________________________                                              NK631 monosulfate                                                             (Cu-free)     155.0      133.6-179.8                                    Rat   RS-form       150.6      129.0-170.6                                          Bleomycin complex                                                                           168.0      130.0-217.0                                    ______________________________________                                    

4-2. Subacute and chronic toxicity in rats and dogs

NK631 monosulfate (copper-free form) was comparable to Bleomycin complexin the subacute and chronic toxicity in rats and dogs. Further, specialmention should be made of the fact that necrosis of injected site wasobserved in all cases of dogs administered with a high dose of Bleomycincomplex (1.2 mg/kg 90 injections), but in none of the cases of dogsadministered with identical dose of NK631 monosulfate (copper-freeform). Toxicity of NK631 monosulfate (copper-free form) to the lung wasalso lower than that of the Bleomycin complex.

From the above results, toxicity of NK631 monosulfate (copper-free form)is comparable to that of Bleomycin complex, except for the toxicity tolung and the necrosis.

5. Summary

From the test results described above, it is concluded that NK631 is anovel compound having the characteristics of:

(1) an extremely low toxicity to the lung;

(2) an antimicrobial and antitumor activity superior to that of thecommercial bleomycin complex;

(3) a systemic toxicity comparable to that of the commercial bleomycincomplex; and

(4) a low local toxicity to the injected site.

Accordingly, NK631 is expected to be useful in clinical fields.

The novel bleomycin of this invention, NK631, is synthesized by reactinga reactive derivative of the carboxyl group of bleomycinic acidrepresented by the formula ##STR2## withN-[(S)-1'-phenylethyl]-1,3-diaminopropane. More particularly, it can beprepared by (1) reacting bleomycinic acid withN-[(S)-1'-phenylethylamino]-1,3-diaminopropane in the presence of anactivating reagent, or (2) reacting bleomycinic acid 3-aminopropyl esteror its N-monosubstituted derivative withN-[(S)-1'-phenylethyl]-1,3-diaminopropane. Detailed description of theseprocedures is given below.

The bleomycinic acid used in the procedure (1) is a known compoundobtained by enzymatic cleavage of bleomycin B₂ according to the methoddisclosed in U.S. Pat. Nos. 3,843,448 and 3,846,400.

Another starting material, i.e.N-[(S)-1'-phenylethyl]-1,3-diaminopropane, is a novel compound firstsynthesized by the present inventors in the following manner.

Phenylethylamine cooled at about 0° C. is admixed with an approximatelyequivalent amount of acrylonitrile. The mixture is kept at 80° to 100°C. for 10 to 24 hours to complete the reaction. The excess acrylonitrileis then removed by distillation under reduced pressure and the residueis further distilled to obtain 3-[(S)-1'-phenylethylamino]propionitrile.The compound thus obtained is reduced in a customary manner, forexample, in the presence of Raney nickel to yield the intendedN-[(S)-1'-phenylethyl-1,3-diaminopropane] (hereinafter referred to as"amino compound"). Physico-chemical properties of this compound are aslisted in Table 7.

                                      Table 7                                     __________________________________________________________________________                         Free base   Dihydrochloride                              __________________________________________________________________________    (1)  Appearance      colorless liquid at                                                                       white needle crystal                                              room temp.                                               (2)  Melting point   --          223.5°-224° C.                 (3)  Boiling point   95°-103° C. (2 mmHg)                                                        --                                           (4)  Ultraviolet absorption                                                                        241, 247, 252, 257,                                                                       250, 256, 259, 266                                maxima (mμ)  263, 267    (distilled water)                                                 (methanol)                                               (5)  Molecular extinction co-                                                                      --          206 (distil. water)                               efficient, ε (256 mμ)                                         (6)  Specific rotation (α).sub.D.sup.25                                                      --          -20.6° (c = 1, distil.                                                 water)                                            D.sub.D.sup.26  -56.38° (undiluted,                                                                --                                                                l = 1)                                                   (7)  Rf value in TL-chromato-                                                      graphy, n-propanol-pyridine-                                                  acetic acid-water                                                                             0.73        0.73                                              (15:10:3:12 V/V),                                                             Avicel-SF®                                                           (8)  Rm value in high voltage                                                      electropheresis, formic                                                       acid-acetic acid-water                                                        (25:75:900 V/V),                                                                              1.52        1.52                                              Avicel-SF®, 800 V,                                                        10 min., Rm of alanine                                                        = 1.0                                                                    (9)  IR absorption spectrum,                                                                       700, 760, 820,                                                                            690, 750, 820,                                    cm.sup.-1, KBr  910, 1,025, 1,080                                                                         910, 980, 1,020,                                                  1,130, 1,200, 1,305,                                                                      1,065, 1,075, 1,150,                                              1,350, 1,370, 1,450,                                                                      1,205, 1,385, 1,460,                                              1,495, 1,605, 2,850,                                                                      1,500, 1,515, 1,590,                                              2,940, 3,300, 3,375                                                                       2,500, 2,850, 3,000,                                                          3,500                                        (10) Molecular formula                                                                             C.sub.11 H.sub.18 N.sub.2                                                                 C.sub.11 H.sub.20 N.sub.2 Cl.sub.2                (molecular weight)                                                                            (178.28)    (251.20)                                     __________________________________________________________________________

Examples of the useful activating reagents include6-chloro-1-p-chlorobenzenesulfonyloxybenzotriazole (CCBT),N-ethyl-5-phenylisoxazolium-3'-sulfonate (NEPIS),N-tert-butyl-5-methylisoxazolium perchlorate,N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, di-p-nitrophenylsulfite, tri-p-nitrophenyl phosphite, p-nitrophenyl trichloroacetate,N-hydroxysuccinimide, dicyclohexylcarbodiimide (DCC),1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide,1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide, diphenylcarbodiimide,di-p-toluylcarbodiimide, diisopropylcarbodiimide, p-nitrophenol,pentachlorophenol, and benzyl alcohol.

Detailed description of the procedure (1):

Bleomycinic acid (copper-containing form) is dissolved in water,dimethylformamide, dimethyl sulfoxide, or a mixture thereof. To thesolution, while being stirred at 0° to 30° C., is added one of theactivating reagents listed above. The resulting solution is adjusted topH 3-10, suitable for activation, by the addition of an inorganic acidor base such as hydrochloric acid or sodium hydroxide or the addition ofan organic acid or base such as trichloroacetic acid orN-methylmorpholine. Upon such a treatment, activation of the carboxylgroup of bleomycinic acid is initiated. Immediately or within 30 minutesafter the treatment, the reactant solution is admixed with the aminocompound (hereinbefore defined) as such or after having been adjusted topH about 7.0. The mixture is then kept at 0° to 30° C. for 1 to 24 hoursto allow the reaction to proceed, thereby yielding NK631. A suitableproportion of the activating reagent or the amino compound is 1 to 10equivalents for 1 equivalent of bleomycinic acid.

In order to isolate NK631 from the reaction mixture, at first thebleomycin compounds are exhaustively precipitated by the addition of 5to 10 volumes of acetone for 1 volume of the reaction mixture. Theprecipitate is collected by filtration, washed with acetone, anddissolved in a least possible quantity of distilled water. The resultingaqueous solution is immediately adjusted to pH 6.0 with hydrochloricacid or the like and applied on the top of a chromatographic columncontaining CM-Sephadex® C-25 (NH₄ ⁺ type, supplied by Pharmacia FineChemicals.) packed in an aqueous ammonium chloride solution. Theunreacted bleomycinic acid passes through the column without beingadsorbed. The NK631 adsorbed on the resin is eluted with aqueous ammoniachloride solutions of gradient concentrations, increasing stepwise orcontinuously from 0.05 M to 1.0 M. NK631 is contained in blue effluentfractions, at about 0.35 M to 0.45 M of ammonium chloride concentration,which show ultraviolet absorption at 292 mμ. These fractions areseparately collected and passed through Amberite® XAD-2 (Rohm and HaasCo.) or Diaion® HP40 (Mitsubishi Chemical Co.). The adsorbed NK631 iswashed with water and eluted to give a desalted effluent, from which apure, blue, amorphous powder of NK631 (copper-containing form) isobtained. Hydrochloride and sulfate of NK631 are obtained by elutingwith aqueous methanol containing hydrochloric acid and sulfuric acid,respectively. In this process, unreacted bleomycinic acid can beconveniently recovered. The procedural step described above, whereinCM-Sephadex® C-25 (a tradename for an cation-exchange Sephadex composedof microscopic beads of carboxymethyl groups derivative frompolysaccharide dextran, manufactured and sold by Pharmacia FineChemicals Inc., Sweden) is employed, is an illustrative example and canbe modified to some degree without substantially affecting the results.For instance, aqueous chloride or aqueous ammonium sulfate can be usedas eluent.

The removal of copper from the copper-containing NK631 obtained abovecan be effected by applying any of the known methods in which copper isremoved by reacting with hydrogen sulfide to convert the copper intocupric sulfide which is precipitated; by reducing the copper tozero-valent copper with a reducing agent (U.S. Pat. No. 3,646,197); byextracting copper with an organic solvent containing a chelating agentsuch as dithizone [Umezawa et al., Journal of Antibiotics, 19A, 210(1960)]; or by the use of a non-ionic-exchange macroreticular resin(U.S. Pat. No. 3,929,993). An example of the copper-removing procedureis described below.

NK631 (copper-containing form) is dissolved in distilled water and thesolution is poured into a resin column containing Amberlite® XAD-2 (atradename for an absorbent resin composed of a styrene-divinylbenzeneco-polymer manufactured and sold by Rohome & Haas Co., U.S.A.) orDiaion® HP40 (a tradename for an absorbent resin composed of astyrene-divinylbenzene co-polymer manufactured and sold by MitsubishiChemical Co., Japan) packed in distilled water to allow the NK631 to beadsorbed. The column is then washed with a 5% aqueous solution ofethylenediaminetetraacetic acid disodium (hereinafter referred to as"EDTA.Na₂ "), whereby the copper ion is carried away by the EDTA.Na₂solution, leaving behind copper-free NK631 on the resin. The resin iswashed with an aqueous solution of a salt such as sodium chloride,sodium sulfate or sodium acetate to remove EDTA.Na₂, and then withdistilled water. Finally, an acidified methanol-water mixture such as,for example, a mixture of methanol and 0.0025 N hydrochloric acid (1:1V/V) is passed through the column to elute a substance showing anultraviolet absorption at 290 mμ. This fraction is collected,concentrated, adjusted to pH 6.0, and freeze-dried to yield apale-yellowish white amorphous powder of NK631 dihyrochloride(copper-free form).

As an acid to be used to prepare an acidified methanol-water mixture,any acids can be used so long as they are pharmaceutically acceptable.For example, when sulfuric acid or acetic acid is used, a powder ofNK631 monosulfate or diacetic acid salt (copper-free form) is obtained.

The KN631 of this invention can be converted in a customary way intoother non-toxic salts such as, for example, sulfate and acetic saltwhich can be also obtained by changing arbitrarily the acid employed ineluting.

In the procedure (1), since the amino compound reacts with the activatedcarboxyl group of bleomycinic acid owing to the presence of anactivating reagent employed in the polypeptide synthesis, thecondensation reaction proceeds smoothly under extremely mild conditions.Consequently, only the primary amino group of the amino compound reactswith the activated carboxyl group while the secondary imino group doesnot participate in the reaction, thus yielding preferentially NK631.This is a remarkable advantage of the procedure (1).

Detailed description of the procedure (2):

The 3-aminopropyl ester of bleomycinic acid employed in the procedure(2) is easily obtained in the form of dihydrochloride (copper-containingform) by thermally decomposing bleomycin A₂ to form3-(methylmercapto)propylaminobleomycin and reacting the resultantbleomycin with a halogenonitrile, halogenoacetic acid, halogenoacetateester, or halogenoacetamide in an acidic solution (U.S. Pat. No.3,886,133).

The N-monosubstituted derivative of 3-aminopropyl ester of bleomycinicacid, which has its amino group protected, is obtained in a high yieldby dissolving dihydrochloride of 3-aminopropyl ester of bleomycinic acidin water or an organic solvent such as methanol or a mixture thereof,then adding slowly to the resulting solution, while stirring vigorously,an equivalent amount or a slight excess of one of the known aminogroup-protecting reagents, in the form of powder or solution in anorganic solvent such as methanol, and allowing the reaction to proceedat room temperature or ice-cooled temperature while continually keepingpH of the reactant mixture at 5.0 to 7.5 by adding an organic base suchas, for example, trimethylamine, triethylamine, pyridine,1,3-diazabicyclo-[5,4,0]-7-undecene, 1,5-diazabicyclo-[3,4,0]-5-nonene,1,4-diazabicyclo[2,2,2]-octane, or N-methylmorpholine. Typical of suchN-monosubstituted derivatives are monohydrochloride of3-acetylaminopropyl ester of bleomycinic acid, 3-succinylaminopropylester of bleomycinic acid, monohydrochloride of 3-benzoylaminopropylester of bleomycinic acid, monohydrochloride of3-benzyloxycarbonylaminopropyl ester of bleomycinic acid,monohydrochloride of 3-p-toluenesulfonylaminopropyl ester of bleomycinicacid, monohydrochloride of 3-(2,4-dinitrophenyl)aminopropyl ester ofbleomycinic acid, monohydrochloride of3-(3,5-dimethyl-3-oxocyclohexen-1-yl)aminopropyl ester of bleomycinicacid, monohydrochloride of 3-N-tert-butoxycarbonylpropyl ester ofbleomycinic acid, and monohydrochloride of 3-N-salicylideneiminopropylester of bleomycinic acid (U.S. Pat. No. 3,886,133).

In the procedure (2), NK631 is formed by the reaction between3-aminopropyl ester or N-monosubstituted 3-aminopropyl ester ofbleomycinic acid and the amino compound. In the case ofN-monosubstituted 3-aminopropyl ester of bleomycinic acid as startingmaterial, either a pure material or a reaction mixture, which may beconcentrated, obtained from 3-aminopropyl ester of bleomycinic acid andan amino group-protecting reagent can be used.

Preferable solvents used in the reaction of 3-aminopropyl ester orN-monosubstituted 3-aminopropyl ester of bleomycinic acid and the aminocompound are water and organic solvents such as methanol,dimethylformamide and dimethyl sulfoxide. The reactant mixture is leftstanding at 0° to 80° C. for 1 to 72 hours under a neutral or alkalinecondition to allow the aminolysis reaction to proceed, whereby NK631 isformed. Under the condition of a higher pH, a prolonged reaction time atcomparatively low temperatures is desirable, while under the conditionof a lower pH, a short reaction time at higher temperatures ispreferred. The suitable proportion of the amino compound in the reactantmixture is 1 to 10 equivalents for 1 equivalent of the 3-aminopropylester of bleomycinic acid or a N-monosubstituted derivative thereof.

To isolate NK631 from the reaction mixture, at first the bleomycincomponents are exhaustively precipitated by the addition of 2 to 5 timesthe volume of reaction mixture of acetone. The precipitate is collectedby filtration, washed thoroughly with acetone and then dissolved in theleast possible amount of distilled water. The resulting aqueous solutionis quickly adjusted to pH 6.0 by adding hydrochloric acid or the likeand poured into a chromatographic column of CM-Sephadex® C-25 (NH₄ ⁺type, supplied by Pharmacia Fine Chemicals) packed in a 0.05 M aqueousammonium chloride solution to allow the bleomycin components to beadosrbed on the resin. Upon passing through the column aqueous ammoniumchloride solution in which the concentration is increased stepwise orcontinually from 0.05 M to 1.0 M, the unreacted 3-aminopropyl ester ofbleomycinic acid or a N-monosubstituted derivative thereof and NK631 areeluted, forming blue bands (UV absorption at 292 mμ) in the effluentstream at ammonium chloride concentrations of 0.15-0.20 M and 0.35-0.45M, respectively. These fractions are separately collected and desaltedby adsorbing on Amberlite® XAD-2 or Diaion® HP40, washing with water,and eluting to obtain a blue amorphous powder of NK631(copper-containing form). Hydrochloride and sulfate are obtained byusing methanol-water mixtures acidified with hydrochloric acid andsulfuric acid, respectively, as eluents. The unreacted 3-aminopropylester of bleomycinic acid or a N-monosubstituted derivative thereof maybe conveniently recovered. The procedural step described above, whereinCM-Sephadex® C-25 is employed, is an typical illustrative example andcan be modified to some degree without substantially affecting theresults. For instance, aqueous sodium chloride or aqueous ammoniumsulfate can be used as the eluent.

The NK631 thus obtained by the procedure (2) can be converted to thecopper-free form by the application of known copper-removing methodsdescribed above in connection with the procedure (1). If necessary, itis possible to convert it into other non-toxic salts such as, forexample, hydrochloride, sulfate and acetic acid salt.

Principal physicochemical properties of NK631 are as shown in Tables 8and 9.

The structural formula (I) of NK631 was confirmed in the following way:NK631 monosulfate (copper-free form) was dissolved in heavy water andmeasured for ¹³ C-NMR by the proton noise decoupling method usingdioxane as internal standard. Signals due to a total of eleven ¹³ Catoms contained in the amino compound with side chain and3-[(S)-1'-phenylethylamino]propylamino moiety were recognized at 19.4,26.3, 37.0, 43.5, 58.9, 128.3 (two signals), 130.0 (two signals) and136.3 ppm. Signals due to other carbon atoms were all corresponded tothe signals common to the bleomycin family [Naganawa et al., Journal ofAntibiotics, 30, 388 (1977)].

                                      Table 8                                     __________________________________________________________________________    Physicochemical properties of NK631                                           (copper-containing form)                                                                      Dihydrochloride                                                                             Monosulfate                                     __________________________________________________________________________    (1)                                                                             Appearance    Blue amorphous powder                                                                       Blue amorphous powder                           (2)                                                                             Solubility    Soluble in water, methanol,                                                                 The same as dihydro-                                            dimethyl sulfoxide, di-                                                                     chloride                                                        methylformamide; sparingly                                                    soluble in dioxane; in-                                                       soluble in ethanol, acetone,                                                  ether, benzene.                                               (3)                                                                             Melting point (decomp.),                                                      °C.    205-207       205-207                                         (4)                                                                             Specific rotation (dis-                                                       tilled water, C = 1.0)                                                        (α).sub.436.sup.25                                                                    -95.6° -93.7°                                   (5)                                                                             TL-chromatography,.sup. 1                                                     R.sub.f value (a) 0.72 (b) 0.75                                                                           (a) 0.72 (b) 0.75                               (6)                                                                             Electrophoresis,.sup. 2                                                       Rm value (alanine = 1)                                                                      0.80          0.80                                            __________________________________________________________________________     Note:                                                                         .sup.1  (a) Silica Gel G® (a tradename for an adsorbent for thin laye     chromatography composed of silica gel manufactured by Merck Inc., U.S.A.)     methanol/10% ammonium acetate/10% ammonia (10:9:1 V/V)                         (b) Avicel SF® (a tradename for an adsorbent for thinlayer               chromatography composed of crystalline cellulose manufactured by FMC          corporation, U.S.A.); npropanol/pyridine/acetic acid/water (15:10:3:12)       .sup.2 Avicel SF®; formic acid/acetic acid/water (25:75:900 V/V); 800     V; 15 minutes                                                            

                                      Table 9                                     __________________________________________________________________________    Physicochemical properties of NK631                                           (copper-free form)                                                                          Dihydrochloride                                                                        Monosulfate                                                                            Di(acetic acid) salt                          __________________________________________________________________________    (1) Appearance                                                                              Pale yellowish                                                                         Pale yellowish                                                                         Pale yellowish                                              white amorphous                                                                        white amorphous                                                                        white amorphous                                             powder   powder   powder                                        (2) Solubility                                                                              Soluble in water, methanol, acetic acid, DMSO, DMF;                           sparingly soluble in dioxane; insoluble in ethanol,                           acetone, ether, benzene                                         (3) Melting point (decomp.),                                                                195-197  196-198  188-190                                        °C.                                                                   (4) Specific rotation (distil.                                                 water, c = 1.0)                                                               (α).sub.436.sup.25                                                                   -2.1°                                                                           -2.0°                                                                           -2.0°                                  (5) TL-chromatography,.sup.1                                                   R.sub.f value                                                                              (a) 0.56 (b) 0.70                                                                      (a) 0.56 (b) 0.70                                                                      (a) 0.56 (b) 0.70                             (6) Electrophoresis,.sup.2                                                     R.sub.m value (alanine = 1.0)                                                              0.94     0.94     0.94                                          __________________________________________________________________________     Note:                                                                         .sup.1 (a) Silica Gel G®; methanol/10% ammonium acetate/10% ammonia       (10:9:1 V/V) (b) Avicel SF®; npropanol/pyridine/acetic acid/water         (15:10:3:12)                                                                  .sup.2 Avicel SF®; formic acid/acetic acid/water (25:75:900 V/V); 800     V; 15 minutes                                                            

In the accompanying drawings, FIGS. 1 and 2 show ultraviolet absorptioncurves of the bleomycin NK631 of this invention and FIGS. 3 and 4 showinfrared absorption curves of NK631.

That is, FIG. 1 shows ultraviolet absorption curve of3-[(S)-1'-phenylethylamino]propylaminobleomycin dihydrochloride(copper-containing form), FIG. 2 shows ultraviolet absorption curve of3[(S)-1'-phenylethylamino]-propylaminobleomycin monsulfate (copper-freeform), FIG. 3 shows infrared absorption curve of3-[(S)-1'-phenylethylamino]propylaminobleomycin dihydrochloride(copper-containing form) measured in the form of potassium bromidetablet, and FIG. 4 shows infrared absorption curve of3-[(S)-1'-phenylethylamino]propylaminobleomycin (copper-free form)measured in the form of potassium bromide tablet.

The invention is illustrated below in detail with reference to Examples,but the invention is not limited to the Examples.

EXAMPLE 1 Synthesis of dihydrochloride (copper-containing form) andmonosulfate (copper-free form) of NK631

Step A: In 400 ml of dimethylformamide, was dissolved 15.0 g ofbleomycinic acid (copper-containing form). To the solution, while beingkept at 0° C. by cooling, were added 1.1 ml of N-methylmorpholine and10.3 g of CCBT. The mixture was stirred for 5 minutes at 0° C., thenadmixed with 5.3 g of the amino compound and further stirred for 1 hour.After termination of the reaction by adding 200 ml of a 25% aqueousacetic acid solution, the reaction mixture was mixed with 5 liters ofcold acetone to precipitate the reaction product. The precipitate wascollected by filtration, washed with acetone, and dissolved in 500 ml ofdistilled water. The resulting aqueous solution was immediately adjustedto pH 6.0 and poured into a column containing 2 liters of CM-Sephadex®C-25 (NH₄ ⁺ type) packed in 0.05 M aqueous ammonium chloride solution toadsorb bleomycins.

Using aqueous ammonium chloride solution, elution was performed bypassing through the column 20 liters of eluent in which theconcentration of ammonium chloride was continually increased from 0.05to 1.0 M. The unreacted bleomycinic acid was found in the effluent atthe ammonium chloride concentration of about 0.05 M and NK631 at theammonium chloride concentration of about 0.45 M. Both fractions, whichshowed UV absorption at 292 mμ, were separately collected. TheNK631-containing fraction was poured into a resin column containing 2.6liters of Amberlite® XAD-2. The column was then washed thoroughly withwater and eluted with 0.01 N hydrochloric acid in methanol-water (4:1V/V). A total of 2.5 liters of the blue fraction, which showed UVabsorption at 292 mμ, was collected. After evaporating off the methanolfrom the eluent fraction, the concentrate was adjusted to pH 6.0 withDowex® 44 (OH⁻ type), (a tradename for an anion-exchange resin composedof a co-polymer of epichlorohydrin and ammonia manufactured and sold byDow Chemical Co., U.S.A.), and was freeze-dried to obtain 16.1 g (9%yield) of NK631 dihydrochloride (copper-containing form) in the form ofblue amorphous powder.

By similar treatment, 280 mg of the unreacted bleomycinic acid(copper-containing form) were recovered.

The ultraviolet absorption maxima and antimicrobial potency of the NK631dihydrochloride (copper-containing form) were as shown below.

UV absorption maxima:

mμ: (E₁ cm^(1%), distilled water)

242: (151)

292: (121)

Antimicrobial potency: 8,100 u/mg

[Note: The antimicrobial potency was assayed using Mycobacteriumsmegmatis ATCC 607 as assay organism and assuming the potency ofbleomycin A₂ (copper-free form) as 1,000 u/mg. The same applieshereinafter.]

Step B: In 200 ml of distilled water, was dissolved 10.0 g of the NK631dihydrochloride (copper-containing form). The solution was poured into acolumn containing 600 ml of Amberlite® XAD-2 packed in distilled water.The column was then washed successively with 2 liters of an aqueoussolution containing 5% of EDTA.Na₂, 2.5 liters of a 5% aqueous sodiumsulfate solution, and 630 ml of distilled water. The column was theneluted with 0.0025 N sulfuric acid in methanol-water mixture (1:1 V/V).A total of 900 ml of fractions containing a substance which showed UVabsorption at 290 mμ was collected. After removal of methanol bydistillation, the residual liquid was adjusted to pH 6.0 with Dowex® 44(OH⁻ type) and freeze-dried to obtain 9.3 g (95% yield) of NK631monosulfate (copper-free form) in the form of pale yellowish whiteamorphous powder. This product showed an UV absorption maximum and anantimicrobial potency as shown below.

UV absorption maximum:

mμ: (E₁ cm^(1%) 0.1 N HCl)

290: (106)

Antimicrobial potency: 7,865 u/mg

EXAMPLE 2 Synthesis of NK631 dihydrochloride (copper-containing form)

In 40 ml of distilled water, was dissolved 1.5 g of bleomycinic acid(copper-containing form). To the stirred solution at 26° C., was added800 mg of NEPIS while maintaining pH of the reactant mixture at 4.8 to5.5 by the addition of 0.1 N aqueous sodium hydroxide solution. After 30minutes, to the mixture was added a solution prepared by dissolving 1.8g of the amino compound in 20 ml of distilled water and adjusting to pH7.0 with hydrochloric acid. The mixture was left standing for 20 hoursat 26° C. To the reaction mixture was added with stirring 600 ml of coldacetone to precipitate the bleomycin components. The precipitate wascollected by filtration, washed with acetone, then dissolved in 30 ml ofdistilled water and immediately adjusted to pH 6.0 with 0.1 Nhydrochloric acid. The resulting aqueous solution was purified byfollowing the procedure described in step A of Example 1 to obtain 932mg (53% yield) of NK631 dihydrochloride (copper-containing form) in theform of blue amorphous powder having an amtimicrobial potency of 8,073u/mg.

EXAMPLE 3 Synthesis of NK631 dihydrochloride (copper-containing form)

In 40 ml of dimethyl sulfoxide, was dissolved 1.5 g of bleomycinic acid(copper-containing form). To the stirred solution at 30° C., were added740 mg of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline and 0.1 ml ofN-methylmorpholine. To the mixture, which had been stirred for 30minutes, was added with stirring 530 mg of the amino compound and themixture was left standing for 3 hours at 30° C. To the reaction mixturewas added with stirring 600 ml of cold acetone to precipitate bleomycincomponents. The precipitate was collected by filtration, washed withacetone, then dissolved in 30 ml of distilled water and immediatelyadjusted to pH 6.0 with 0.1 N hydrochloric acid. The resulting aqueoussolution was treated in a manner similar to that in step A of Example 1to obtain 809 mg (46% yield) of NK631 dihydrochloride (copper-containingform) in the form of blue amorphous powder which showed an antimicrobialpotency of 8,028 u/mg.

EXAMPLE 4 Synthesis of NK631 dihydrochloride (copper-containing form)

In 40 ml of distilled water, was dissolved 1.5 g of bleomycinic acid(copper-containing form). After addition of 1.2 g of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride at 27° C.,the mixture was adjusted to pH 4.5 with 0.1 N hydrochloric acid. Afteraddition of 530 mg of the amino compound, the mixture was again adjustedto pH 5.0 with 1 N hydrochloric acid and left standing for 20 hours at27° C. To the reaction mixture, was added with stirring 400 ml of coldacetone to precipitate the bleomycin components. The precipitate wascollected by filtration, washed with acetone, then dissolved in 30 ml ofdistilled water and immediately adjusted to pH 6.0 with 0.1 N sodiumhydroxide. The resulting aqueous solution was purified by following theprocedure described in step A of Example 1 to obtain 440 mg (25% yield)of NK631 dihydrochloride (copper-containing form) in the form of blueamorphous powder having an antimicrobial potency of 7,985 u/mg.

EXAMPLE 5 Synthesis of NK631 dihydrochloride (copper-containing form)

In 16 ml of methanol, was dissolved 9.0 g of bleomycinic acid3-N-benzoylaminopropyl ester monohydrochloride (copper-containing form).While cooling at 0° C., to the solution was added 9.16 g of the aminocompound. After continued stirring for 40 hours at 0° C., cold acetonewas added to the reaction mixture to precipitate the reaction products.The precipitate was collected by filtration, washed with acetone, anddissolved in 300 ml of distilled water. The solution was immediatelyadjusted to pH 6.0 with 0.1 N hydrochloric acid and poured into a columncontaining 1 liter of CM-Sephadex® C-25 (NH₄ ⁺ type) packed in 0.05 Maqueous ammonium chloride solution to allow the bleomycins to beadsorbed. Using aqueous ammonium chloride solution as eluent, elutionwas performed by passing through the column 10 liters of eluent in whichthe concentration of ammonium chloride was continuously increased from0.05 to 1.0 M. The unreacted 3-N-benzoylaminopropyl ester of bleomycinicacid was found in the effluent at the ammonium chloride concentration ofabout 0.2 M and NK631 at the ammonium chloride concentration of about0.45 M. Both fractions, which showed UV absorption at 272 mμ, wereseparately collected. The NK631-containing fraction was poured into aresin column containing 1.3 liters of Amberlite® XAD-2. The column wasthen washed thoroughly with water and eluted with 0.01 N hydrochloricacid in methanol-water mixture (4:1 V/V). A total of 2.5 liters of theblue fraction, which showed UV absorption at 292 mμ, was collected.After having been freed from methanol by distillation and adjusted to pH6.0 with Dowex® 44 (OH⁻ type), the fraction was freeze-dried to obtain8.2 g (90% yield) of NK631 dihydrochloride (copper-containing form) inthe form of blue amorphous powder having an antimicrobial potency of8,030 u/mg.

By similar treatment, 910 mg of the unreacted bleomycinic acid3-N-benzoylaminopropyl ester monohydrochloride (copper-containing form)were recovered.

EXAMPLE 6 Synthesis of NK631 dihydrochloride (copper-containing form)

In 3 ml of methanol, was dissolved 950 mg of bleomycinic acid3-N-p-toluenesulfonylaminopropyl ester monohydrochloride(copper-containing form). To the solution, while being stirred at 0° C.,was added 980 mg of the amino compound and the mixture was stirred for48 hours at 0° C. To the reaction mixture, was added with stirring 15 mlof cold acetone to precipitate bleomycin components. The precipitate wascollected by filtration, washed with acetone, then dissolved in 30 ml ofdistilled water, and immediately adjusted to pH 6.0 with 0.1 Nhydrochloric acid. The resulting solution was purified following theprocedure described in step A of Example 5 to obtain 833 mg (85% yield)of NK631 dihydrochloride (copper-containing form) in the form of blueamorphous powder having an antimicrobial potency of 8,010 u/mg.

EXAMPLE 7 Synthesis of NK631 dihydrochloride (copper-containing form)

In 5 ml of dimethylformamide, was dissolved 850 mg of bleomycinic acid3-acetylaminopropyl ester monohydrochloride (copper-containing form). Tothe solution, while being stirred at 0° C., was added 950 mg of theamino compound and the mixture was stirred for 45 hours at 0° C. To thereaction mixture, was added with stirring 20 ml of cold acetone toprecipitate bleomycin components. The precipitate was collected byfiltration, washed with acetone, then dissolved in 30 ml of distilledwater, and immediately adjusted to pH 6.0 with 0.1 N hydrochloric acid.The resulting solution was treated in the same manner as in step A ofExample 5 to obtain 570 mg (63% yield) of NK631 dihydrochloride(copper-containing form) in the form of blue amorphous powder having anantimicrobial potency of 8,010 u/mg.

EXAMPLE 8 Synthesis of NK631 dihydrochloride (copper-containing form)

In 3 ml of dimethyl sulfoxide, was dissolved 900 mg of bleomycinic acid3-N-(2,4-dinitrophenyl)-aminopropyl ester monohydrochloride(copper-containing form). To the solution, while being stirred at 0° C.,was added 793 mg of the amino compound and the mixture was stirred for24 hours at 0° C. To the reaction mixture, was added with stirring 15 mlof cold acetone to precipitate bleomycin components. The precipitate wascollected by filtration, washed with acetone, then dissolved in 30 ml ofdistilled water, and immediately adjusted to pH 6.0 with 0.1 Nhydrochloric acid. The resulting aqueous solution was purified byfollowing the procedure described in step A of Example 5 to obtain 690mg (78% yield) of NK631 dihydrochloride (copper-containing form) in theform of blue amorphous powder having an antimicrobial potency of 8,010u/mg.

EXAMPLE 9 Synthesis of NK631 dihydrochloride (copper-containing form)and copper-free dihydrochloride

In 10 ml of methanol, was dissolved 1.0 g of bleomycinic acid3-aminopropyl ester dihydrochloride (copper-containing form). To thevigorously stirred solution, was added dropwise 104 mg of benzoylchloride over a period of 30 minutes while maintaining pH of thesolution at 6.5 to 7.5 by the addition of 1,4-diazabicyclo[2,2,2]octane.After stirring for additional 30 minutes, the reaction mixture wasconcentrated to 2 ml. To the concentrated material cooled at 0° C., wasadded with stirring 1.1 g of the amino compound and the mixture wasstirred for 72 hours at 0° C. To the reaction mixture, was added withstirring 6 ml of cold acetone to precipitate the bleomycin components.The precipitate was collected by filtration, washed with acetone, thendissolved in 30 ml of distilled water, and immediately adjusted to pH6.0 with 0.1 N hydrochloric acid. The resulting aqueous solution waspoured into a column containing 100 ml of CM-Sephadex® (NH₄ ⁺ type)packed in 0.05 M aqueous ammonium chloride solution to allow thebleomycin components to be adsorbed. The adsorbed material was treatedin a manner similar to that of step A in Example 5 to obtain 779 mg (73%yield) of NK631 dihydrochloride (copper-containing form) in the form ofblue amorphous powder having an antimicrobial potency of 8,000 u/mg.

In addition, 240 mg of the unreacted bleomycic acid3-N-benzoylaminopropyl ester monohydrochloride (copper-containing form)were recovered.

Step B: In 20 ml of distilled water, was dissolved 700 mg of the NK631dihydrochloride (copper-containing form) obtained in step A above. Thesolution was poured into a column containing 65 ml of Diaion® HP-40packed in distilled water to allow the bleomycin components to beadsorbed. The column was then washed with 200 ml of an aqueous solutioncontaining 5% of EDTA.Na₂, then with 250 ml of a 5% aqueous sodiumchloride solution, and finally with 100 ml of distilled water. Thecolumn was then eluted with a mixture of methanol and 0.0025 N aqueoushydrochloric acid (1:1 V/V) and 98 ml of the fraction showing ultraviletabsorption at 290 mμ were collected. After removal of the methanol bydistillation under reduced pressure, the residual liquid was adjusted topH 6.0 with Dowex® 44 (OH⁻ type) and freeze-dried to obtain 658 mg (98%yield) of NK631 dihydrochloride (copper-free form) in the form of paleyellowish white amorphous powder having an antimicrobial potency of7,834 u/mg.

EXAMPLE 10 Synthesis of NK631 monosulfate (copper-containing form) anddiacetic acid salt (copper-free form)

Step A: In 10 ml of methanol, was dissolved 1.0 g of bleomycinic acid3-aminopropyl ester dihydrochloride (copper-containing form). To thevigorously stirred solution at room temperature, was added dropwise 104mg of benzoyl chloride over a period of 30 minutes while maintaining pHof the solution at 5.0 to 7.5 by the addition of N-methylmorpholine.After stirring for additional 30 minutes, to the reaction mixture wasadded 50 ml of acetone to precipitate the bleomycin components. Theprecipitate was collected by filtration, washed with acetone and againdissolved in 3 ml of methanol. To the methanol solution, while beingcooled at 0° C. and stirred, was added 597 mg of the amino compound andthe mixture was stirred for 42 hours at 0° C. To the reaction mixture,was added with stirring 10 ml of cold acetone to precipitate thereaction product. The precipitate was collected by filtration, washedwith acetone, then dissolved in 30 ml of distilled water and immediatelyadjusted to pH 6.0 with 0.1 N hydrochloric acid. The aqueous solutionwas treated with CM-Sephadex® in a manner similar to that in step A ofExample 5. The fraction containing the reaction products was poured intoa column containing 65 ml of Diaion® HP-40 packed in distilled water toallow the bleomycin components to be adsorbed on the resin. Afterwashing with water, the adsorbed phase was eluted with a mixture ofmethanol and 0.01 N aqueous sulfuric acid (1:1 V/V) and 130 ml of afraction showing ultraviolet absorption at 292 mμ were collected. Afterremoval of methanol by distillation, the residual liquid was adjusted topH 6.0 with Dowex® 44 (OH⁻ type) and freeze-dried to obtain 813 mg (75%yield) of NK631 monosulfate (copper-containing form) in the form of blueamorphous powder having an antimicrobial potency of 7,819 u/mg.

Step B: Copper-removing treatment of 800 mg of the NK631 monosulfate(copper-containing form) obtained in step A above was carried out in thesame manner as in step B of Example 9, except that in the elution stagea 5% aqueous ammonium acetate solution and a mixture of methanol and0.01 N aqueous acetic acid (1:1 V/V) were used in place of the 5%aqueous sodium chloride solution and the mixture of methanol and 0.0025N aqueous hydrochloric acid (1:1 V/V), respectively. There were thusobtained 748 mg (96% yield) of NK631 diacetic acid salt (copper-freeform) in the form of pale yellowish white amorphous powder. The UVabsorption maximum and the antimicrobial potency of this product were asshown below.

UV absorption maximum:

mμ: (E₁ ^(1%) _(cm) 0.1 HCl)

290: (105)

Antimicrobial potency: 7,620 u/mg

EXAMPLE 11 Synthesis of NK631 dihydrochloride (copper-containing form)and copper-free dihydrochloride

In 3 ml of methanol, was dissolved 1.0 g of bleomycinic acid3-aminopropyl ester dihydrochloride. To the solution, was added dropwiseat room temperature 193 mg ofS-tert-butoxycarbonyl-4,6-dimethyl-2-mercaptopyrimidine over a period of60 minutes, while maintaining pH at 6.0 to 7.4 by the addition of a 14%triethylamine in methanol. The mixture was stirred for further 2 hoursto yield 3N-tert-butoxycarbonylaminopropyl ester of bleomycinic acid.The mixture was then cooled to 0° C., admixed with 1.2 g of the aminocompound and stirred for 70 hours. To the reaction mixture was added 10ml of cold acetone to precipitate the bleomycin components. Theprecipitate was collected by filtration, washed with acetone, thendissolved in 30 ml of distilled water, and immediately adjusted to pH6.0 with 0.1 N hydrochloric acid. The resulting aqueous solution waspurified by following the procedure used in step A of Example 5 toobtain 597 mg (56% yield) of NK631 dihydrochloride (copper-containingform) in the form of blue amorphous powder having an antimicrobialpotency of 8,010 u/mg.

Step B: In 50 ml of methanol, was dissolved 500 mg of NK631dihydrochloride (copper-containing form) obtained in step A above. Intothe vigorously stirred solution, was introduced hydrogen sulfide througha nozzle, 1 mm in internal diameter and dipped into the solution, for 1hour to dissolve a large excess of hydrogen sulfide in the solution.After termination of the introduction of hydrogen sulfide, the solutionwas left standing for 30 minutes at room temperature. The precipitatedcopper sulfide was collected by filtration and washed with 50 ml ofmethanol saturated with hydrogen sulfide. The filtrate and washings werecombined and freed from the methanol and hydrogen sulfide bydistillation under reduced pressure. The residue was dissolved in 50 mlof methanol and mixed with 100 ml of ethyl ether. The precipitate formedwas collected by filtration, washed with ether and dried to obtain 394mg (82% yield) of NK631 dihydrochloride (copper-free form) in the formof pale yellowish white amorphous powder having an antimicrobial potencyof 7,820 u/mg.

EXAMPLE 12 Synthesis of NK631 dihydrochloride (copper-containing form)and copper-free dihydrochloride

Step A: In 2 ml of methanol, was dissolved 1.0 g of bleomycinic acid3-aminopropyl ester dihydrochloride (copper-containing form). To thevigorously stirred solution at room temperature, was added 98 mg ofsalicyaldehyde while maintaining pH of the solution at 7.0 to 7.4 by theaddition of triethylamine to yield bleomycinic acid3-N-salicylideneiminopropyl ester. After 1 hour of continued stirring,to the mixture cooled to 0° C., was added with stirring 600 mg of theamino compound. The mixture was stirred for 30 hours. To the reactionmixture was added 10 ml of cold acetone to precipitate the bleomycincomponents. The precipitate was collected by filtration, washed withacetone, then dissolved in 30 ml of distilled water, and immediatelyadjusted to pH 6.0 with 0.1 N hydrochloric acid. The resulting aqueoussolution was purified as in step A of Example 5 to obtain 501 mg (47%yield) of NK631 dihydrochloride (copper-containing form) having anantimicrobial potency of 8,013 u/mg.

Step B: In 25 ml of 0.5 N aqueous hydrochloric acid, was dissolved 450mg of the NK631 dihydrochloride (copper-containing form) obtained instep A above. To the solution was added 25 ml of a chloroform solutioncontaining 0.2% of dithizon (diphenylthiocarbazone). After rapid shakingand mixing, the mixture was allowed to stand still, whereby the mixtureseparated into two layers. The lower layer (chloroform layer) was drawnoff and the upper layer was mixed with 25 ml of the fresh chloroformsolution containing dithizon. The above procedure of shaking, phaseseparation, and addition of fresh chloroform solution containingdithizon was repeated eight times. Finally the upper layer (aqueouslayer) was washed with chloroform and the separated aqueous layer wasadjusted to pH 6.0 with Dowex® 44 (OH⁻ type). The aqueous layer thustreated was evaporated under reduced pressure to dryness to obtain 415mg (96% yield) of NK631 dihydrochloride (copper-free form) in the formof pale yellowish white amorphous powder having an antimicrobial potencyof 7,845 u/mg.

EXAMPLE 13 Synthesis of NK631 dihydrochloride (copper-containing form)

In 2 ml of methanol, was dissolved 1.0 g of bleomycinic acid3-aminopropyl ester dihydrochloride (copper-containing form). To thesolution cooled to 0° C., was added with stirring 1.0 g of the aminocompound and the mixture was stirred for further 72 hours. To thereaction mixture, was added with stirring 6 ml of cold acetone toprecipitate the bleomycin components. The precipitate was purified as instep A of Example 5 to obtain 128 mg (12% yield) of NK631dihydrochloride (copper-containing form) in the form of blue amorphouspowder having an antimicrobial potency of 8,015 u/mg.

REFERENCE EXAMPLE 1 Synthesis ofN-[(S)-1'-phenylethyl]-1,3-diaminopropane

To 50 g of (S)-1-phenylethylamine at 0° C. with cooling, was added withstirring 33 g of acrylonitrile. The mixture was placed in a flaskprovided with a reflux condenser connected to a calcium chloride tubeand was heated to 93° C. at which temperature the mixture was stirredfor 18 hours. After completion of the reaction, the reaction mixture wasfreed from the excess acrylonitrile by distillation under reducedpressure. The residue was distilled under reduced pressure and afraction boiling at 142° to 145° C./7 mmHg was collected to obtain 53.5g of 3-[(S)-1'-phenylethylamino]propionitrile. This nitrile was placedin an autoclave together with 5 g of Raney nickel W-7 and 50 ml ofethanol containing 15% of ammonia. The mixture was stirred at a speed of1,000 rpm for 1.5 hours, at 50° to 58° C., and under a hydrogen partialpressure of 100 to 40 kg/cm². After completion of the reduction, thereaction mixture was distilled under reduced pressure. A fractionboiling at 95°-103° C./2 mmHg was collected to obtain 48.3 g ofN-[(S)-1'-phenylethyl]-1,3-diaminopropane [66% theoretical yield from(S)-1-phenylethylamine].

What is claimed is:
 1. A method for producing3-[(S)-1'-phenylethyl-amino] propylaminobleomycin represented by theformula ##STR3## and a non-toxic salt thereof, which comprises reactinga reactive derivative of the carboxylic group of the bleomycinic acidrepresented by the formula ##STR4##
 2. A method according to claim 1,wherein the non-toxic salt is hydrochloride, sulfate or acetic acidsalt.
 3. A method according to claim 1, wherein 1 to 10 equivalents ofthe N-[(S)-1'-phenylethyl]-1,3-diaminopropane are used for oneequivalent of the bleomycinic acid.
 4. A method according to claim 1,wherein the bleomycinic acid and theN-[(S)-1'-phenylethyl]-1,3-diaminopropane are allowed to react at 0° to30° C. for 1 to 24 hours.
 5. A method according to claim 1, wherein thesolvent is dimethylformamide, diethylsulfoxide or a mixture thereof.